The melanocortin 1 receptor gene (MC1R) is a main contributor to the diversity of human pigmentation. The activated MC1R stimulates eumelanin synthesis, and enhances nucleotide excision repair and antioxidant responses in UV-irradiated human melanocytes, thus reducing the risk for melanoma. Functional variants of MC1R disrupt receptor function and alter the pigmentary phenotype by varying extents. We propose to investigate the hypothesis that MC1R functional variants increase melanoma risk by disrupting MC1R signaling, leading to impaired UV-induced DNA damage response of human melanocytes via compromising their DNA repair, antioxidant and melanogenic capacities. The effects of most MC1R variants on the function of the melanocyte are poorly understood. Given the lack of an appropriate animal model for human pigmentation, the unique properties of the human MC1R, and the role of human melanocytes in photoprotection, it is critical to investigate the impact and establish causality of common human MC1R variants in the aberrant UV response, using human melanocytes that naturally express this gene and are a physiological target for melanocortins. A unique resource available to us is a cohort of patients with known MC1R genotype, pigmentary phenotype, and clinical history and risk factors for melanoma, which will allow us to correlate the MC1R genotype with phenotype. Melanocyte cultures will be established from skin biopsies taken from these patients. A major strength of this application is the complementary expertise of the Principal Investigators, Dr. Abdel-Malek, a pioneer in the research on melanocortins and human pigmentation, and Dr. Leachman, an expert in the molecular, genetic, and clinical aspects of melanoma, and the unique expertise of the co-Investigators and collaborators. Three Specific Aims are proposed to link the function of the MC1R to its main signaling pathway, the cAMP pathway, and to the UV-induced MAP kinase pathway, which regulates critical biological endpoints that determine melanomagenesis, namely DNA repair, antioxidant defenses and melanogenesis. In Specific Aim 1, we will compare how specific MC1R variants affect MC1R function by reducing ligand binding and/or signaling by failure to increase cAMP synthesis that activates the main signaling pathway for melanocortins. In Specific Aim 2, we will investigate in UV-irradiated melanocytes the impact of different MC1R variants on the MAP kinases and three of their downstream transcription factors NRF2, ATF2 and USF-1 that regulate the expression of antioxidant genes, DNA repair and apoptosis genes, and the pigmentary genes POMC, MC1R, and tyrosinase, respectively. In Specific Aim 3, we will determine how different MC1R variants expressed in human melanocytes affect the UV-induced levels and repair rates of cyclobutane pyrimidine dimers and 8-hydroxy-deoxyguanosine, generation of reactive oxygen species, and stimulation of melanogenesis in cultured melanocytes and skin substitutes. These studies will improve risk assessment of melanoma susceptible individuals and development of effective strategies for melanoma prevention and early detection.